There are no FDA approved drugs for the prevention and treatment of acute kidney injury (AKI). Thus, mortality remains high and survivors of AKI suffer from significant morbidity; many develop chronic kidney disease and end stage renal disease. A precise understanding of early mechanisms of AKI is critically needed to guide development of novel therapies. Over the past two grant cycles, we identified novel therapeutic targets for the prevention and treatment of AKI. Among these, tissue resident dendritic cells (DCs) play disparate roles in tissue homeostasis - assuming a central role in the early pathogenesis of AKI, yet also playing a tolerogenic role - and in different forms of AKI, where they promote injury in ischemia-reperfusion injury (IRI) and are protective in cisplatin nephrotoxicity (CN). During the current funding cycle new information has shown that DCs can initiate immune responses following kidney IRI by activating natural killer T cells via the IL23/IL17 pathway. Abrogating this process, e.g. by adenosine 2a receptor (A2aR) agonist administration, attenuates injury. DCs express A2aRs; we demonstrate that DCs are tolerized by exposure to A2aR agonists. We propose studies to better understand the role of DC function in AKI and the role of adenosine signaling through endogenous DC A2aRs in two models of AKI (IRI and CN). These fundamental findings form the basis of a cell-based therapeutic approach, modeled after current clinical trials in cancer, autoimmune diseases, and transplantation, to tolerize DCs ex vivo with A2aR agonists for the prevention and treatment of AKI. Our preliminary data demonstrate that endogenous DC A2aRs critically control the extent of IRI, administration of A2a-tolDCs (up to 7 days before or 6 hrs after injury) protects kidneys from IRI, A2a agonists block CN, and allogenic A2a-tolDCs block IRI. Our studies will define the mechanism by which A2a agonists tolerize both murine and human DCs and the mechanism by which they prevent kidney AKI in vivo. The significance of this approach is that cell-based therapy using A2a-tolDC promotes superior tissue protection with a broad therapeutic window, avoids systemic administration of a drug (or other drugs) that could potentially have adverse side effects such as platelet dysfunction, hypotension and systemic immunosuppression, serves as a proof of concept model that tolerized DCs (from any treatment modality) may be useful in future strategies for the treatment of AKI, and promotes prolonged protection due to a lasting memory effect of A2a agonists on DCs. Using transgenic mice, selective pharmacological reagents, and novel reagents, we will test the hypotheses that (Aim 1) endogenous DC A2aRs control the extent of IRI and CN, (Aim 2) A2a-tolerized DCs are effective in prevention of AKI and treatment of established AKI, and (Aim 3) A2a-tolDCs protect kidneys by enhancing IL-10 signaling and regulatory T cells. These studies will form the basis of therapy for prevention of AKI and more broadly for transplantation and other autoimmune diseases.